家蚕尿苷磷酸化酶1的克隆与表达分析

Cloning and expression of silkworm(Bombyx mori)uridine phosphorylase 1

【目的】通过对家蚕尿苷磷酸化酶1基因(BmUPase1)的克隆、原核表达、生物信息学分析及各组织和各时期的表达变化的研究,为揭示其在家蚕Bombyx mori中发挥的作用奠定基础。【方法】以家蚕整蚕的cDNA为模板克隆获得BmUPase1基因的编码框序列。利用生物信息学软件分析序列特性。对该基因编码的蛋白质进行原核表达,为后续制备抗体提供条件。通过qPCR对BmUPase1在家蚕各组织中和各时期的表达量进行分析。【结果】BmUPase1基因的完整ORF框序列全长1188 bp,编码395个氨基酸,分子式为C1945H3074N5380591S21,分子量大小为44.12 ku,具有尿苷磷酸化酶(UPase)超家族的保守结构域PNP_UDP_1。进化分析可知:家蚕与斜纹夜蛾Spodoptera litura、棉铃虫Helicoverpa armigera、小菜蛾Plutella xylostella、夏威夷红蛱蝶Vanessa tameamea等亲缘关系最近。qPCR结果显示BmUPase1基因在家蚕各个组织中都有表达,说明BmUPase1对于家蚕的生长发育具有重要的作用。时期表达谱结果显示,BmUPase1在家蚕化蛹、蛹期和蛾期的表达量升高,推测其与家蚕化蛹及化蛾有关。pET28a-BmUPase1重组载体在大肠杆菌BL21中成功表达,与预测大小一致。【结论】本研究首次克隆了BmUPase1基因,其在家蚕的各个组织和各个时期都有表达,可能对家蚕的生长发育具有重要的作用。对其表达模式进行分析,为进一步研究蛋白质的功能奠定基础。

[Objectives]To investigate the characteristics of uridine phosphorylase in the silkworm by cloning,prokaryotic expression,bioinformatics analysis,and thereby lay a foundation for elucidating the molecular mechanism of UPase in this economically important species.[Methods]The CDS of BmUPase1 was cloned using Dazao cDNA and analyzed with bioinformatic software.The gene was then inserted into the prokaryotic expression vector pET28 a to create a recombinant plasmid pET28 a-BmUPase1,which was then transformed into Escherichia coli BL21(DE3)to express BmUPase1.Real-time quantitative PCR was performed to profile the expression of the gene in different tissues and growth stages.[Results]Bioinformatics analysis showed that the BmUPase1 gene has an ORF of 1188 bp and encodes 395 amino acids.BmUPase1 has one conserved domain,and its amino acid sequence identity is most similar to that of Spodoptera litura UPase(85.06%).qPCR results indicate that the BmUPase1 gene is expressed in all tissues and growth stages,which suggests that it plays an important role in silkworm growth and development.The pET28 a-BmUPase1 recombinant vector was successfully expressed in E.coli BL 21,and the expressed protein was of the predicted size.[Conclusion]The silkworm Uridine phosphorylase gene can be successfully cloned and expressed.The results of this study lay a foundation for further study of the function of this gene in silkworm development.